AB0092 CTLA4-Ig PROMOTES THE M1-M2 SHIFT IN CULTURED MACROPHAGES OF RHEUMATOID ARTHRITIS PATIENTS WITH ACTIVE DISEASE: IN VITRO STUDY
Authors: M. Cutolo, S. Tardito, E. Gotelli, P. Montagna, R. Campitiello, S. Paolino, C. Pizzorni, A. Sulli, V. Smith, S. Soldano
Journal: Annals of the Rheumatic Diseases (2022)

Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarization into “classically” (M1) or “alternatively activated” (M2) phenotypes and the release of pro-inflammatory cytokines (1). In the active inflammatory phase of RA, circulating intermediate monocytes and synovial tissue macrophages show a M1 phenotype, whereas MerTK + M2 macrophages seem to characterize the synovial tissue of RA patients under remission (2-4). In RA, CTLA4-Ig fusion protein (abatacept) reduces the pro-inflammatory activity of macrophages by interacting with the costimulatory molecule CD86 on surface cell membrane of activated cells, including macrophages (2). Objectives The in vitro study investigated the efficacy of CTLA4-Ig treatment to induce the shift from the M1 phenotype into an M2 phenotype in cultured monocyte-derived macrophages (MDMs) obtained from active RA patients. Methods Cultured MDMs obtained from peripheral blood mononuclear cells of 5 active RA patients (mean age 54±13 years) and 5 age-matched healthy subjects (HSs) after overnight stimulation with phorbol myristate acetate (5ng/ml), were treated with CTLA4-Ig at the concentrations of 100mg/mL or 500mg/mL for 3, 12, 24 and 48 hours. A part of cultured RA-MDMs as wells as cultured HS-MDMs were maintained in growth medium (RPMI at 10% of fetal bovine serum) without any treatment and used as unstimulated cells. Gene expression of CD80, CD86 and toll-like receptor-4 (TLR4), as M1 markers, as well as macrophage scavenger receptors (CD163, CD204), mannose receptor-1 (CD206), as surface M2 markers, and MerTK (functional M2 marker) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by Wilcoxon t-test. Results Cultured RA-MDMs showed a high basal gene expression of TLR4, CD80 and CD86 compared to HS-MDMs, confirming to be activated M1 macrophages. In these macrophages, CTLA4-Ig treatment downregulated the gene expression of M1 markers at both concentrations and all timings, but significantly limited to TLR4 and CD80 markers (500mg/mL,12 hours: p&lt;0.05). Conversely, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD163, MerTK and CD206 (p&lt;0.05), whereas only the high concentration of CTLA4-Ig significantly upregulated CD204 gene expression (p&lt;0.05). The protein synthesis of all M2 surface markers was increased after 24 hours of treatment primarily by the high concentration of CTLA4-Ig, and significantly for CD204 and CD206 (p&lt;0.05). Conclusion CTLA4-Ig treatment seems to exert an important anti-inflammatory effect by promoting the shift from a M1 to an M2 phenotype in cultured RA macrophages The results suggest a further mechanism for CTLA4-Ig in the modulation of the RA synovitis (5). References [1]Yang X et al. Cell Prolif. 2020;53:e12854.doi:10.111/cpr.12854. [2]Kumar RA et al. Int. Immunol.2018;65:348-59. [3]Boutet MA et al. Autoimmun Rev.2021;20:102758. doi: 10.1016/j.autrev.2021.102758. [4]Alivernini S et al. Nat Med. 2020;26:1295-306. 5. Cutolo M et al. Arthritis Res Ther. 2009;11:R176; doi: 10.1186/ar2865. Disclosure of Interests Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Celgene, Pfizer, Boehringer Ingelheim, Samuele Tardito: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Rosanna Campitiello: None declared, Sabrina Paolino: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratories Baldacci, Vanessa Smith Grant/research support from: Boehringer Ingelheim, Janssen-Cilag, Stefano Soldano: None declared